Recombinant lactic acid bacteria and the use thereof in oral universal influenza vaccine

Abstract

The present invention relates to an oral universal influenza vaccine comprising recombinant lactic acid bacteria which express proteins including but not limited to ferritin protein plus highly-conserved stem fragment of hemagglutinin (HA) proteins expressed in all known influenza viruses. The present invention also relates to the recombinant protein comprising the highly-conserved stem fragment of HA and ferritin proteins.

Claims

1. An oral formulation for targeting a variety of influenza viruses causing influenza and treating said influenza in a subject in need thereof comprising a recombinant lactic acid bacterial cell transformed with an expression vector containing an encoding sequence for encoding a fusion protein comprising a fluorescent protein, a stem domain of influenza hemagglutinin, a ferritin protein and two linkers, and a pharmaceutically acceptable carrier, wherein the encoding sequence encoding the fluorescent protein and encoding the stem domain of influenza hemagglutinin have been codon optimized for expression in lactic acid bacteria and wherein in the fusion protein said two linkers link the stem domain of influenza hemagglutinin, the fluorescent protein, and the ferritin protein.

2. The oral formulation of claim 1, wherein said fluorescent protein is orange fluorescent protein originated from Cerianthus sp.

3. The oral formulation of claim 1, wherein said stem domain of influenza hemagglutinin comprises a H1HA10 stem fragment and T4 bacteriophage fibritin foldon.

4. The oral formulation of claim 1, wherein said ferritin protein is originated from Helicobacter pylori.

5. The oral formulation of claim 1, wherein said encoding sequence is one of the SEQ ID NOs: 1-6.

6. The oral formulation of claim 5, wherein said encoding sequence is SEQ ID NO: 1.

7. The oral formulation of claim 1, wherein said lactic acid bacterial cell is from the strains comprising Lactobacillus sp. and Lactococcus sp.

8. The oral formulation of claim 1, wherein said expression vector is an expression vector that is for expressing protein in gram-positive bacterial host cells.

9. The oral formulation of claim 1, wherein said lactic acid bacterial cell is from Lactobacillus casei.

10. The oral formulation of claim 1, wherein said expression vector is pTRKH3.

11. The oral formulation of claim 1, wherein the encoding sequence for the first linker is positioned between the encoding sequence of the fluorescent protein and stem domain of influenza hemagglutinin, and the encoding sequence for the second linker is positioned between the encoding sequence of the stem domain of influenza hemagglutinin and ferritin protein.

12. The oral formulation of claim 11, wherein said first linker comprises an Factor Xa (Xa) protease encoding sequence, a restriction site of KpnI, and a poly-His encoding sequence.

13. The oral formulation of claim 11, wherein said second linker comprises Tobacco Etch Virus (TEV) protease encoding sequence and a restriction site of AgeI.

14. A method for treating influenza caused by a variety of influenza viruses comprising orally administering the oral formulation of claim 1 to a subject in need thereof.

15. The method of claim 14, wherein said oral formulation is orally administered to said subject once daily for three consecutive days on weekly basis and for two consecutive weeks.

16. The oral formulation of claim 1, wherein said variety of influenza viruses comprises H1, H3, and H5.

17. The oral formulation of claim 1, wherein said oral formulation is formulated into a form comprising powder, pills, capsules, liquid, and tablets.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a schematic diagram depicting the design of the DNA insert into an expression vector for transformation into a Lactobacillus strain according to a preferred embodiment of the present invention: Region 1 represents a modified DNA sequence of orange fluorescent protein (OFP); Region 2 represents a modified DNA sequence of influenza HA stem fragment; Region 3 represents a DNA sequence of ferritin; two small brackets represent two linkers for linking up the DNA sequences of the OFP and influenza HA stem fragment and those of the influenza HA stem fragment and the ferritin, respectively.

(2) FIG. 2 shows the result of an agarose gel analysis of Lactobacillus casei transformed clones with an expression vector containing the DNA insert depicted in FIG. 1: (M) DNA marker: (L) PCR amplification from transformed Lactobacillus casei clone for clone confirmation.

(3) FIG. 3 shows the result of an agarose gel analysis of restriction enzyme digested cloned plasmid isolated from transformed Lactobacillus casei clones according to an embodiment of the present invention: (M) DNA marker; (1-4) digested cloned plasmid from Lactobacillus casei transformed clones.

(4) FIG. 4 shows the result of Western blot analysis of influenza hemagglutinin stem fragment-containing recombinant protein (OFP-HIHA10-Foldon-Ferritin) extracted from transformed Lactobacillus casei according to an embodiment of the present invention: (C) proteins extracted from wild-type Lactobacillus casei which serves as control: (1) and (2) proteins extracted from transformed Lactobacillus casei.

(5) FIG. 5 shows the result of Western blot analysis of mouse serum two weeks after oral administration of the transformed Lactobacillus casei: (1) PBS only; (2) wild-type Lactobacillus casei (110.sup.9 cfu per mouse); and (3) the transformed Lactobacillus casei (110.sup.9 cfu per mouse) according to an embodiment of the present invention, respectively.

(6) FIG. 6 is a schematic diagram showing a structure of influenza hemagglutinin.

(7) FIG. 7 shows the plasmid construction of the expression vector with the desired DNA insert for transformation into Lactobacillus casei according to a preferred embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

(8) References in the specification to one embodiment, an embodiment, an example embodiment, etc., indicate that the embodiment described can include a particular feature, structure, or characteristic, but every embodiment may not necessarily include the particular feature, structure, or characteristic. Moreover, such phrases are not necessarily referring to the same embodiment. Further, when a particular feature, structure, or characteristic is described in connection with an embodiment, it is submitted that it is within the knowledge of one skilled in the art to affect such feature, structure, or characteristic in connection with other embodiments whether or not explicitly described.

(9) Values expressed in a range format should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a concentration range of about 0.1% to about 5% should be interpreted to include not only the explicitly recited concentration of about 0.1 wt. % to about 5 wt. %, but also the individual concentrations (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, and 3.3% to 4.4%) within the indicated range.

(10) As described herein, the terms a or an are used to include one or more than one and the term or is used to refer to a nonexclusive or unless otherwise indicated. In addition, it is to be understood that the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description only and not of limitation. Furthermore, all publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference. In the event of inconsistent usages between this document and those documents so incorporated by reference, the usage in the incorporated reference should be considered supplementary to that of this document; for irreconcilable inconsistencies, the usage in this document controls.

(11) In the methods of manufacturing described herein, the steps can be carried out in any order without departing from the principles of the invention, except when a temporal or operational sequence is explicitly recited. Recitation in a claim to the effect that first a step is performed, and then several other steps are subsequently performed, shall be taken to mean that the first step is performed before any of the other steps, but the other steps can be performed in any suitable sequence, unless a sequence is further recited within the other steps. For example, claim elements that recite Step A, Step B, Step C, Step D, and Step E shall be construed to mean step A is carried out first, step E is carried out last, and steps B, C, and D can be carried out in any sequence between steps A and E, and that the sequence still falls within the literal scope of the claimed process. A given step or sub-set of steps can also be repeated.

(12) Furthermore, specified steps can be carried out concurrently unless explicit claim language recites that they be carried out separately. For example, a claimed step of doing X and a claimed step of doing Y can be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process.

Definitions

(13) The singular forms a,, an and the can include plural referents unless the context clearly dictates otherwise.

(14) The term about can allow for a degree of variability in a value or range, for example, within 10%, or within 5% of a stated value or of a stated limit of a range.

(15) The term independently selected from refers to referenced groups being the same, different, or a mixture thereof, unless the context clearly indicates otherwise. Thus, under this definition, the phrase X1, X2, and X3 are independently selected from noble gases would include the scenario where, for example, X1, X2, and X3 are all the same, where X1, X2, and X3 are all different, where X1 and X2 are the same but X3 is different, and other analogous permutations.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

(16) The present invention is not to be limited in scope by any of the following descriptions. The following examples or embodiments are presented for exemplification only.

EXAMPLES

(17) The embodiments of the present invention can be better understood by reference to the following examples which are offered by way of illustration. The present invention is not limited to the examples given herein.

Example 1Design of DNA Insert and Transformation of Expression Vector Containing the Same in Host Cell

(18) FIG. 1 shows the design of DNA insert comprising a modified orange fluorescent protein (OFP) encoding sequence (Region 1). The sequence is originated from Cerianthus sp. (GenBank sequence database: AAP55761.1). The protein expressed from this modified OFP encoding sequence can enhance the immunogenicity of the recombinant protein of the present invention in the recipient. The modified encoding sequence of OFP also matches the codon preference of LAB in general. However, other commercially available fluorescent protein having the foregoing characteristics can also be used in the DNA insert.

(19) The DNA insert of FIG. 1 also comprises a modified influenza hernagglutinin stem fragment encoding sequence (Region 2), which is referenced to protein sequences disclosed in Mallajosyula et al. (Mallajosyula, Vamsee V A. et al. Proceedings of the National Academy of Sciences 111.25 (2014): E2514-E2523). Differing from the disclosed sequences in Mallajosyula et al., reverse translation from one of the disclosed sequences followed by codon optimization to Lactobacillus caseiare done, and some modifications are madein the DNA sequence after reverse translation for optimization purposes. A T4 bacteriophage fibritin foldon is also encoded in the underlined sequence in Region 2 as shown in FIG. 1.

(20) The DNA insert of FIG. 1 further comprises an encoding sequence of ferritin protein (Region 3), which is originated from Helicobacter pylori (NCBI Reference Sequence Database: WP_000949190.1).

(21) Between Region 1 and Region 2, the DNA insert of FIG. 1 also comprises a first linker which is a poly-His tag linker. The first linker comprises an Xa protease encoding sequence and a restriction site of KpnI, before the poly-His encoding sequence.

(22) Between Region 2 and Region 3, the DNA insert of FIG. 1 further comprises a second linker. The second linker comprises TEV protease encoding sequence and a restriction site of AgeI.

(23) The sequence of the DNA insert of FIG. 1 is also represented by SEQ ID NO: 1. Optionally, a sequence comprising at least one restriction site can be inserted before and/or after the start codon and stop codon of the DNA insert. For example, NcoI and NdeI restriction sites can be inserted before the start codon; BamHI restriction site can be inserted after stop codon.

(24) The DNA sequence as shown in FIG. 1 (or SEQ ID NO: 1) is cloned in the pTRKH3 expression vector for protein expression in the transformed Lactobacillus casei. FIG. 7 shows the plasmid construction map of said pTRKH3 expression vector inserted with the DNA encoding sequence of SEQ ID NO: 1.

(25) It should be understood that the order of different regions in the DNA insert as described in the present invention can be changed, provided that the expressed proteins from the Lactobacillus casei transformed with said expression vector with these different DNA inserts having different combinations of said regions are capable of inducing antibody against the variety of H subtype influenza viruses in the subject that receives the formulation comprising the transformed Lactobacillus casei. For example, these different inserts may have the following combinations of different regions: (i) Region 1 (OFP) is followed by Region 3 (ferritin protein) and then Region 2 (stem domain of influenza HA), which encoding sequence is represented by SEQ ID NO: 2; (ii) Region 2 (stem domain of influenza HA) is followed by Region 1 (OFP) and then Region 3 (ferritin protein), which encoding sequence is represented by SEQ ID NO: 3; (iii) Region 2 (stem domain of influenza HA) is followed by Region 3 (ferritin protein) and then Region 1 (OFP), which encoding sequence is represented by SEQ ID NO: 4; (iv) Region 3 (ferritin protein) is followed by Region 1 (OFP) and then Region 2 (stem domain of influenza HA), which encoding sequence is represented by SEQ ID NO: 5; (v) Region 3 (ferritin protein) is followed by Region 2 (stem domain of influenza HA) and then Region 1 (OFP), which encoding sequence is represented by SEQ ID No: 6.
It is also possible to only include the encoding sequence of Region 2 into the expression vector, from which the protein expressed can still induce antibody against said variety of H subtype influenza viruses in said subject.

(26) To confirm positive clones. PCR amplification is employed to screen and identify Lactobacillus casei successfully transformed with pTRKH3 expression vector containing the DNA insert of SEQ ID NO: 1, namely OFP-H1HA10-Foldon-Ferritin encoding sequence. In FIG. 2, PCR amplification products from positive clones are analyzed by gel electrophoresis in 1% agarose gel. A pair of forward and reverse primers are used for the amplification, namely SEQ ID NO: 8 and SEQ ID NO: 9. The expected length of the PCR product is 1,766 bp, in which the corresponding band on the gel from the lane loaded with the PCR product is between 1,650 bp and 2,000 bp with respect to the DNA marker next to the lane of the PCR product. The result indicates that the expression vector, pTRKH3, containing the DNA insert of SEQ ID NO: 1 comprising said modified OFP encoding sequence, modified influenza HA stem fragment encoding sequence and ferritin protein encoding sequence has high transformation efficiency. Positive clones so screened are subject to further confirmation and studies.

(27) The DNA encoding sequence of influenza hemagglutinin stem fragment (H1HA10-Foldon) is isolated from the expression vector extracted from the transformed positive clones confirmed in FIG. 2. Under double-digestion by using two specific restriction enzymes (KpnI and AgeI), the digested expression vector samples extracted from the positive clones are analyzed by agarose gel (1%) electrophoresis. FIG. 3 shows that there are two distinct bands in four samples, and it is confirmed that the expression vector used in this example is successfully transformed inside the host bacteria.

Example 2Expression of Recombinant Protein Comprising Influenza HA Stem Fragment

(28) The positive transformed Lactobacillus casei clones from Example 1 which are successfully selected are capable of expressing the influenza hemagglutinin stem-fragment. To further confirm that the corresponding target fragment is expressed in the transformed Lactobacillus casei, Western blot is performed to assess the expression efficiency of the hemagglutinin stem fragment inside the transformed Lactobacillus casei. Positive clones are cultured in MRS broth supplemented with 100 g/ml erythromycine at 37 C. with shaking at 250 rpm in anaerobic conditions for 72 hours.

(29) After that, the bacterial cell suspension is collected and the collected cells are lysed to collect the cytosolic proteins. In FIG. 4, two samples are run in SDS gel followed by Western blot analysis. The significant band indicates the presence of the expression of the influenza hemagglutinin stem fragment from the transformed Lactobacillus casei. The amino acid sequence of the recombinant protein containing the modified OFP, modified influenza HA stem fragment (H1HA10-Foldon) and ferritin protein (OFP-H1HA10-Foldon-Ferritin) is represented by SEQ ID NO: 7.

Example 3In Vivo Study of Immunogenicity of Recombinant Lactic Acid Bacteria

(30) BALB/c mice (6-8 weeks) (n=7) are randomly divided into three groups. Three groups are orally administered with three different formulations: PBS (Group 1), wild-type Lactobacillus casei resuspended in 200 l PBS (110.sup.9 cfu per mouse) (Group 2); and transformed Lactobacillus casei confirmed in Examples 1 and 2 resuspended in 200 l PBS (110.sup.9 cfu per mouse) (Group 3), respectively. Oral gavage is repeated three times (once daily, consecutive three days) on a weekly basis for two consecutive weeks. Mice are boosted twice after one week. Blood samples are collected from tail vein at week 3, 5, 7, 9 and 15 after oral gavage of three different formulations, and stored at 80 C. before testing for the presence of the antibodies interested. Also, the weight of each mouse is recorded weekly during the course of feeding period so as to monitor the health conditions of individual mouse after consumption of Lactobacillus casei. No loss of weight is observed and all mice are alive after the course of oral gavage as described hereinbefore.

(31) The confirmed positive clones of expression vector containing the encoding sequence of OFP-H1HA10-Foldon-Ferritin is transformed into DH50 for generation of antibodies. The antibodies so generated are purified using His-bind fractogel column. The purified antibodies are loaded in SDS-PAGE, probed with mouse antibody from mouse serum and detected with HRP-conjugated goat anti-mouse IgG antibody (1:1000) and visualized with ECL Western blotting substrate. After feeding mice with transformed Lactobacillus casei, mouse serum is collected after two weeks of oral feeding and the antibody against the influenza hemagglutinin stem fragment is detected in mouse serum whereas no corresponding antibody is detected in mice orally fed with PBS or wild-type Lactobacillus casei. These results suggest that mice orally fed with transformed Lactobacillus casei generate the corresponding antibody against hemagglutinin stem fragment.

INDUSTRIAL APPLICABILITY

(32) The present recombinant lactic acid bacteria is useful in manufacturing into a vaccine which is safe to be orally administered into a subject such as human and other animals (e.g., livestock). The expressed recombinant protein isolated and purified from a host transformed with the expression vector containing the encoding sequence of the modified influenza hemagglutinin may be used in manufacturing of the vaccine. The vaccine is not limited to oral formulation but it can also include other forms such as injectable form, provided that it does not induce immune-rejection.

(33) Deposition of Microorganism

(34) Pursuant to the requirements under PCT Rule 13bis, the present modified recombinant Lactobacillus casei has been deposited at the following International Depositary Authority:

(35) Name of Deposit Institute: Guangdong Microbial Culture Collection Center (GDMCC), Guangdong Institute of Microbiology

(36) Address of Deposit Institute: No. 59 Building, 100 Xianlie Central Road, Guangzhou 510070, China

(37) Date of Deposit: 18 Nov. 2016

(38) GDMCC No.: 60113